Tuberculous pleural effusion-induced Arg-1+ macrophage polarization contributes to lung cancer progression via autophagy signaling

Background The association between tuberculous fibrosis and lung cancer development has been reported by some epidemiological and experimental studies; however, its underlying mechanisms remain unclear, and the role of macrophage (MФ) polarization in cancer progression is unknown. The aim of the present study was to investigate the role of M2 Arg-1+ MФ in tuberculous pleurisy-assisted tumorigenicity in vitro and in vivo. Methods The interactions between tuberculous pleural effusion (TPE)-induced M2 Arg-1+ MФ and A549 lung cancer cells were evaluated. A murine model injected with cancer cells 2 weeks after Mycobacterium bovis bacillus Calmette–Guérin pleural infection was used to validate the involvement of tuberculous fibrosis to tumor invasion. Results Increased CXCL9 and CXCL10 levels of TPE induced M2 Arg-1+ MФ polarization of murine bone marrow-derived MФ. TPE-induced M2 Arg-1+ MФ polarization facilitated lung cancer proliferation via autophagy signaling and E-cadherin signaling in vitro. An inhibitor of arginase-1 targeting M2 Arg-1+ MФ both in vitro and in vivo significantly reduced tuberculous fibrosis-induced metastatic potential of lung cancer and decreased autophagy signaling and E-cadherin expression. Conclusion Tuberculous pleural fibrosis induces M2 Arg-1+ polarization, and M2 Arg-1+ MФ contribute to lung cancer metastasis via autophagy and E-cadherin signaling. Therefore, M2 Arg-1+ tumor associated MФ may be a novel therapeutic target for tuberculous fibrosis-induced lung cancer progression. Supplementary Information The online version contains supplementary material available at 10.1186/s12931-024-02829-8.


Background
Although several epidemiological studies have reported an association between tuberculosis (TB) and cancer development, the underlying mechanisms remain unclear [1][2][3].
The metabolic function and immune properties of macrophages (MФ) in response to M. tuberculosis infection are associated with pathogenicity and outcomes [4].In the late adaptation/resolution phase, a metabolic shift occurs, and increased PGC-1 beta level promotes M2 polarization and inhibits proinflammatory and antimicrobial responses [5].M2 MФ predominate in granulomatous lung tissues compared to M1 MФ [6].Exosomal miRNA from M2 MФ promotes the progression of pulmonary fibrosis [7].
Tumor-associated MФ (TAM) play a pro-tumoral role in the tumor microenvironment by enhancing the rate of tumor cell invasion, extravasation, and growth [8].The immunosuppressive TAM (M2) may inhibit natural killer cells and T cells during tumor progression [9].
In a previous study, we found that the NOX4/ autophagy axis mediates tuberculous fibrosis-induced tumorigenicity [10].Helfinger et al. found that NOX4 regulates MФ polarization [11].Zhang et al. reported that tumoral NOX4-educating M2 MФ promote lung cancer growth [12].Based on these results, we hypothesized that MФ polarization after TB infection contributes to a microenvironment that is susceptible to tumor progression and acts as a new therapeutic target.
In this study, we aimed to examine the effect of tuberculous pleural effusion (TPE)-induced Arg-1 M2 MФ polarization on the invasion of A549 cells using an in vitro co-culture method.We evaluated the treatment efficacy of inhibiting Arg-1 M2 MФ polarization in TB-associated lung cancer in vitro and in vivo using a Mycobacterium bovis bacillus Calmette-Guérin (BCG)induced pleurisy mouse model.

Cell lines and animals
The human adenocarcinoma cell line A549, mouse musculus lung squamous cell line KLN205, and mouse fibroblast cell line L-929 (American Type Culture Collection, Manassas, VA, USA) were cultured according to the manufacturer's instructions.A549 cells were maintained in RPMI 1640 medium (BYLABS, Hanam, Korea) supplemented with 10% FBS, 100 U/mL penicillin, and 100 μg/ mL streptomycin at 37 °C in a humidified incubator with 5% CO 2 .KLN205 cells were maintained in Eagle's Minimum Essential medium (Corning, Manassas, VA, USA) supplemented with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin at 37 °C in a humidified incubator with 5% CO 2 .L-929 cells were maintained in DMEM/ F12 (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% horse serum at 37 °C in a humidified incubator with 5% CO 2 .L-929 cell culture medium (conditioned medium, L-929 culture medium (CM)) was collected and centrifuged at 3,000 rpm and 4 °C, passed through a 0.45-µm filter, and frozen at -27 °C in 50 mL aliquots.

Reagents and kits
Human XL Cytokine Array Kit (R&D Systems, Minneapolis, MN, USA) was used to compare cytokine profiles between TPE and transudate (T).Recombinant CXCL9 and CXCL10 and neutralizing antibodies against CXCL9 and CXCL10 were purchased from R&D Systems.The arginase inhibitor 2(S)-amino-6-boronohexanoic acid (ABH) was purchased from Cayman Chemicals (Ann Arbor, Michigan, USA) and used in cell and mouse experiments.The autophagy inhibitors 3-methyladenine (3-MA) and bafilomycin A1 (BafA1) were purchased from Enzo Life Sciences (Farmingdale, NY, USA).

Bone marrow-derived MФ differentiation and polarization
MФ were derived from the bone marrow cells of 6-7-week-old C57BL/6J mice.The cells were differentiated for 6 days in α-minimum essential medium supplemented with 30% L-929 CM.The bone marrow-derived MФ (BMDMs) were treated with 1:1 dilution of TPE or T for 48 h to induce polarization.Thereafter, the MФ supernatant (TPE-Arg-1 + MФ CM or T-Arg-1-MФ CM) was harvested and used to treat A549 cells (Fig. 1a).

RNA extraction, real-time polymerase chain reaction, western blotting, and enzyme-linked immunosorbent assay
Total RNA was extracted from A549 cells or mouse lung tissue samples using easy-Blue reagent (iNtRON Biotechnology, Seoul, Korea).First-strand cDNA was synthesized using the Maxime RT PreMix Kit (iNtRON Biotechnology) according to the manufacturer's protocol.Real-time polymerase chain reaction (PCR) was performed on Rotor-Gene Q using SYBR Green I as a double-stranded DNA-specific dye according to the manufacturer's instructions.The PCR mixture of total volume 20 μL comprised 1 μL cDNA, 10 μL Power SYBR Green PCR Master Mix (Thermo Fisher Scientific), and 10 pM each of forward and reverse primers.Relative expression was calculated as ddCt, and the data for each sample were normalized to the expression of β-actin mRNA.The sequences of primers used for each gene are presented in Table S1.For immunoblot analysis, the cells and tissues were lysed with RIPA buffer containing a protease inhibitor cocktail (GenDEPOT, Baker, TX, USA).Membranes were incubated overnight at 4 °C with the primary antibodies against actin, LC3B, ATG12, p-Beclin, Beclin, E-cadherin (Cell Signaling Technology, Danvers, MA, USA), ZO-1 (Abcam, Cambridge, UK), p62 (Santa Cruz Biotechnology, Dallas, TX, USA), and Bcl-2 (Enzo Life Sciences).After washing the blots in TBS containing Tween-20, they were incubated with horseradish peroxidase-conjugated secondary antibodies corresponding to each primary antibody.Signals were detected using the ECL detection kit (Thermo Fisher Scientific).Mouse TGF-β or Arginase-1 in serum samples was measured using an ELISA kit (Abbkine, Inc., Atlanta, Georgia, USA), according to the manufacturer's protocol.

Cell invasion assay
A549 cell or MФ invasion assay was performed using Matrigel Transwell chambers with a pore size of 8 μm (Corning).For A549 cell invasion assay, A549 cells were seeded in the upper chamber and the CM of MФ (TPE-Arg-1 + + MФ, T-Arg-1 − + MФ, and ABH + TPE Arg-1 + + MФ) was added to the lower chamber.For the MФ invasion assay, TPE-Arg-1 + + MФ or T-Arg-1 − + MФ were seeded (1 × 10 5 cells/well) in the upper chamber and A549 cells were seeded in the lower chamber.After 24 h, the invasive cells that penetrated the Matrigel barrier to the lower surface were stained with crystal violet solution and counted.

Animal experiments
To develop a BCG-induced pleural fibrosis model, mice were injected with 10 6 colony forming units of BCG Pasteur in 100 µL of PBS into the intrapleural cavity; after 2 weeks, mouse musculus lung squamous cells (KLN205; ATCC) were intravenously injected.ABH (200 μg/mouse) was administered intraperitoneally every other day for 22 days from the day of BCG injection.On day 10 after KLN205 injection, the mice were anesthetized with 4% isoflurane (Piramal Critical Care, Bethlehem, PA, USA), and then lung tissue and serum samples were collected.Paraffin-embedded lung tissue samples were sectioned to 5-μm thickness and stained with hematoxylin and eosin.Histological changes were imaged under a light microscopy (ZEISS Colibri 7, Land Baden-Württemberg, Germany).The lung tissue sections were immunostained with antibodies against collagen (Invitrogen), Arginase-1 (LSBio, Shirley, MA, USA), LC3B (Cell Signaling Technology, Danvers, MA, USA), and F4/80 (Abcam, Cambridge, UK).The samples were analyzed under a fluorescence microscope (Olympus FV500; Olympus, Tokyo, Japan).DAPI (Sigma, St. Louis, MO, USA) was used as a counterstain.

Human samples
The study was approved by the hospital's research ethics committee (institutional review board numbers: 2012-27 and 2017-47).Written informed consent was obtained from all participants before sample collection.Pleural effusion samples were obtained via routine thoracentesis.The definitions of TPE and T were consistent with those of a previous study [13].Pleural effusion samples were collected in sterile tubes and centrifuged at 3,000 g for 10 min and the cell-free supernatant was stored at −80 °C for analysis.

Statistical analyses
All experiments were conducted in triplicate.GraphPad Prism (V.7, GraphPad, USA) was used for statistical analyses.Quantification of lung metastasis lesions were performed using Qupath software (https:// qupath.github.io/).Variables are presented as mean ± standard error of the mean.An unpaired Student's t-test was used to compare the differences between means.Statistical significance was set at p < 0.05.

TPE induces M2 Arg-1 + MФ polarization
Figure 1a shows a schematic representation of the experimental procedure.To evaluate whether TPE-induced Arg-1 + MФ polarization promotes A549 cell growth in vitro, we first treated differentiated MФ with TPE or T for 48 h, and then, MФ CM (Arg-1 + MФ CM or Arg-1 − MФ CM) was added to A549 cells and incubated for 48 h to determine the growth of A549 cells.Subsequently, the A549 cells were analyzed using TEM, real-time PCR, and western blotting.Compared to T, TPE treatment upregulated the expression of M2-associated makers Arg-1 and Ym-1, but did not increase that of the M1-associated markers MCP-1 and iNOS.TPE treatment did not induce cell death (Fig. S1).
To determine the effect of A549 cells on MФ migration, murine MФ were seeded in the upper chambers of Transwell chambers and treated with the CM of A549 cells for 48 h (Fig. S3).The migration rate of TPE-Arg-1 + MФ increased compared with that of the control and T-Arg-1 − MФ groups.These results indicate that the influence of cancer cells on MФ migration increased during TPE treatment, and consequently, the interaction between the cell types was amplified.

CXCL9 and CXCL10 in TPE mediate Arg-1 + MФ polarization
To investigate the TPE components responsible for MФ polarization, we performed a cytokine array analysis of TPE.CXCL9 and CXCL10 expression was upregulated in TPE compared with that in T, whereas apolipoprotein A-1, YKL-40, IGFBP-2, and IGFBP-3 expression was upregulated in T compared with that in TPE (Fig. 3a).To further confirm that CXCL9 and CXCXL10 regulate MФ M2 polarization, we performed chemokine stimulation and neutralization experiments on MФ.Stimulation with 20 µg CXCL9 and 20 µg CXCL10 increased Arg-1 + MФ polarization after 48 h (Fig. 3b).The level of CXCR3, the receptor of CXCL9 and CXCL10, decreased after 24 h, and then replenished at 48 h.
When TPE treatment of MФ was followed by the addition of CXCL9 antibody (4 µg) or CXCL10 antibody (4 µg) for 48 h, the Arg-1 + M2 MФ polarization rate decreased compared with that after only TPE treatment.These results suggest that CXCL9 and CXCL10 elicited by TB development may be involved in M2 MФ polarization.

Arginase inhibition reduces the growth of A549 cells exposed to TPE by suppressing autophagy signaling
We performed experiments to select the concentrations of ABH at which cell viability is maintained and Arg-1 MФ polarization is reduced (Fig. 4a and b).BMDMs were incubated with 100 µg ABH for 24 h, and then with TPE for 48 h.The CM of ABH + TPE-Arg-1 + MФ was used to treat A549 cells for comparison with that of TPE-Arg-1 + MФ.Electron microscopy showed that the number of autophagosomes in the ABH + TPE-Arg-1 + CM group was significantly lower than that in the TPE-Arg-1 + MФ CM group, as confirmed using the Transwell invasion assay and western blotting (Fig. 4c).The ABH + TPE-Arg-1 + CM group showed reduced expression of LC3, ATG12, Beclin, and Bcl-2, but increased expression of P62, compared to the TPE-Arg-1 + MФ CM group (Fig. 4d).In addition, the expression of E-cadherin and ZO-1 was lower in the ABH + TPE-Arg-1 + CM group than in the TPE-Arg-1 + MФ CM group (Fig. 4d).This result shows that ABH may downregulate autophagy and E-cadherin signaling induced by TPE-Arg-1 + MФ as cancer progression pathways.

Effect of ABH in the BCG-induced lung cancer model in vivo
The effects of Arg-1 inhibition on BCG pleurisy-associated lung cancer cells were evaluated in vivo.We treated KLN205 tumor-bearing C57BL6 mice after BCG injection with a commercially available Arg-1 inhibitor (ABH) at a dose of 200 μg i.p. every 2 days for a total of 12 times, and tumor growth rate was monitored over 22 days.Hematoxylin and eosin staining showed that ABH treatment with BCG injection resulted in a significant reduction in the number and size of lung metastatic nodules compared to BCG injection alone (Fig. 5a).
In accordance with the in vitro experimental results, the expression of Arg-1 mRNA expression was upregulated in the BCG + KLN205 group and downregulated in the BCG + ABH + KLN205 group (Fig. 5b).Among the MФ markers, the M2 marker Ym-1 and M1 marker iNOS did not show such a pattern, and MCP-1 and F4/80 levels tended to be lower in the BCG + ABH + KLN205 group than in the BCG + KLN205 group; however, the results were not significant.Similarly, the plasma arginase-1 level in the BCG + KLN205 group was higher than that in the PBS + _KLN205 group and its level in the BCG + ABH + KLN205 group was lower than that in the BCG + KLN205 group (Fig. 5c).The expression of the proinflammatory cytokine TGF-β, related to the progression of cancer, was reduced in the BCG + ABH + KLN205 group compared with that in the BCG + KLN205 group (Fig. 5d).
Arg-1 M2 polarization-autophagy/E-cadherin signaling is involved in TB-related lung cancer progression.Compared to the PBS + KLN205 group, the BCG + KLN205 group mice showed increased expression of ATG12-5, LC3II/LC3I, Beclin, E-cadherin, and ZO-1, but decreased expression of P62.In the BCG + ABH + KLN205 group, the expression of ATG12-5, LC3II/LC3I, Beclin, E-cadherin and ZO-1 decreased, but that of P62 increased, compared with those in the BCG + KLN205 group (Fig. 5f ).Confocal immunofluorescence microscopy was used to confirm these findings.The lung sections of the BCG + KLN205 group had high levels of Arg-1 that colocalized with LC3B (Fig. 6).The inhibition of Arg-1 by ABH treatment attenuated LC3B expression induced by BCG + KLN205.In addition, the distribution of BCG-induced Arg-1 was similar to that of collagen.This finding suggests that Arg-1 MФ are associated with collagen production.

Discussion
The World Health Organization has reported that at least 20% of cancers have an infectious origin [14].Several epidemiological studies have reported an association between TB and cancer development [2,3,15].TB and cancer impose high socio-economic burden and are associated with high mortality rates [16].Both diseases have been extensively researched; however, the overlap between them has not been examined in detail.An experimental study showed that chronic TB in mice could cause lung carcinogenesis by inducing DNA damage and epiregulin expression [17].It has been hypothesized that TB causes lung cancer through genomic injury, cell death evasion, immune system suppression, and oncogene overexpression [16,18].However, robust experimental data are needed to establish a proof of concept.
In our previous study, we found that the NOX4autophagy axis regulated by tuberculous fibrosis could result in enhanced tumorigenic potential [10,16].We hypothesized that after TB infection, the tumor microenvironment changes and becomes susceptible to tumor progression-inducing factors.Among the diverse immune cells that constitute the tumor microenvironment, MФ are particularly abundant and play a role in all stages of tumor progression.TAM can function as either tumoricidal agents (M1) or tumor-promoting immunosuppressants (M2) [19,20].TAMs provide a suitable microenvironment to induce growth, immunosuppression, invasion and chemotherapy resistance in lung cancer [21].TAM secretes pro-angiogenic and tumor-inducing chemokines such as TGF-β, IL-10, CCL18, matrix metalloproteases, epidermal growth factors and TGF-β [21,22].Also, TAM impede the immunoregulatory functions of The CD8+ T cells and tumor infiltrating lung dendritic cells [22][23][24].Previous studies demonstrated the association between TAMs and chemotherapy resistance [25,26].Preclinical experiments have shown that targeting TAMs can be beneficial, as it can inhibit regulatory T-cell mechanisms and enhance the effect of anticancer therapy [9,[27][28][29].During TB development, MФ exhibit phenotypic and functional heterogeneity depending on the site and stage of infection [30].Robust innate immune responses in lung-resident MФ, monocytes, and monocyte-derived MФ play protective roles during TB development [30].Meanwhile, based on the suppressive or hyperactivated immune status, the MTB virulence factor can affect MФ homeostasis and facilitate the development of granulomatous disease and spread of bacilli [31].MTB promotes the M1-M2 switch of MФ to transform from an acute to chronic infection and develop drug resistance [32,33].Especially arginine metabolism plays a role in the differential regulation of MФ polarization [34].Arginase, a pivotal metalloenzyme, metabolizes L-arginine to L-ornithine [35].Thus, arginase plays an important role in fundamental cellular functions, such as wound healing and tissue fibrosis, and suppresses T-cell activation by locally depleting l-arginine [19].
MTB increased Arg-1 expression and substantially reduced bactericidal NO production by competing for l-arginine with i-NOS in a murine model [36].Consistently, Pessanha et al. demonstrated the expression of Arg-1 in the granuloma of tissues samples obtained from patients with TB [37].However, the exact role of Arg-1 in MTB-infected human lungs remains unknown.Some studies have reported the usefulness of L-Arg supplementation in patients with active TB; however, further research on its therapeutic and prophylactic roles is needed [38,39].
In this study, MФ stimulation using tuberculous pleural fluid significantly increased Arg-1 MФ polarization, and similar results were observed in vivo.The cytokine assays and inhibition experiments confirmed that CXCL9 and CXCL10 present in TPE affected MФ Arg-1 polarization.A recent study also demonstrated that the CXCL9-CXCR3 axis plays a role in the migration and activation of MФ in an apical periodontitis model [40].The CXCL9 and CXCL10/CXCR3 axes play two roles in the tumor environment: paracrine signaling for immune activation and autocrine signaling for cancer proliferation and metastasis [41].Similar to a double-edged sword, CXCL9 not only inhibits tumor growth by recruiting CTL, but also contributes to cancer proliferation by recruiting Tregs, TAMs, and MDSCs, which are involved in immune tolerance in tumors [42].
Most importantly, we found that Arg-1 + MФ polarization induced by TPE contributed to lung cancer proliferation by enhancing autophagy signaling and E-cadherin expression.Arg-1 + MФ CM-treated A549 cells showed increased expression of LC3II/LC3I, ATG12-ATG5, Beclin 1, and E-cadherin, and reduced expression of P62.Autophagy has dual effects [43].While cell intrinsic autophagy plays a role in preventing cancer proliferation at the initial stage, under stress conditions, autophagy is associated with tumorigenesis, tumor-stromal interactions, and chemoresistance in the tumor microenvironment [43].Several studies have attempted to improve the anticancer effects of drugs by targeting specific autophagic processes [44,45].
The downregulation of E-cadherin-mediated cell adhesion is related to epithelial-mesenchymal transition in tumor invasiveness [46].On the other hands, E-cadherin plays a role in cancer progression via the PI3K-AKR and MEK-ERK pathways [46].Padmanaban et al. showed that E-cadherin functions as a survival factor in breast cancer by reducing reactive oxygen species-mediated apoptotic signaling [47].Similarly, the previous study demonstrated that high E-cadherin expression in the lung tumor was associated with worse overall survival in patients with stage IV EGFR-mutant lung adenocarcinoma [48].The complicated link between autophagy and EMT exists [49].Autophagy activation can suppress or promotes EMT by regulating various signaling pathways.However, in this study, the increase in E-cadherin in tuberculous fibrosis-induced lung cancer appears to be related to cell basal extrusion, local invasion, distant metastasis, and tumor cell circulation rather than a complete EMT process [50].High levels of autophagy in oncogenic K-ras cells promotes basal extrusion of epithelial cells that is related to tumor promoting functions of E-cadherin [51].
Treatment with an Arg-1 inhibitor reduced the expression of LC3II/LC3I, ATG12, Beclin 1, and E-cadherin and increased the expression of p62 in this study.We found that the Arg-1 inhibitor suppressed cancer progression by modulating autophagy and E-cadherin signaling in TB-associated lung cancer.Based on these findings, TB-associated lung cancer appears to have anticancer drug-resistance properties and specific molecular characteristics.
Previous studies have reported that immunotherapy resistance can be overcome by modulating myeloid cells and TAM [20,52].Further studies on TB-associated lung cancer are needed to confirm chemoresistance and improvement of treatment efficacy when an Arg-I inhibitor is used in combination with other drugs such as immune checkpoint inhibitors.

Conclusions
We found that Arg-1 MФ play an important role in the progression of lung cancer in TB-associated microenvironments.TB-associated lung cancer is associated with autophagy and E-cadherin signaling, which are related to chemoresistance.Our results showed the translational potential of Arg-1 inhibitors for TB-associated lung cancer and demonstrated a challenge for the metabolic regulation of immune responses in both TB and lung cancer.Further research is required to evaluate the chemoresistance and stemness of TB-associated lung cancer, which will ultimately translate into enhanced therapeutics.